Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype

Summary Segments of the TL-DNA of the agropine type Ri plasmid pRi 1855 encompassing single and groups of open-reading frames were cloned in the Ti plasmid-derived binary vector system Bin 19. Leaf disc infections on Nicotiana tabacum led to transformed plants, some of which showed typical hairy root phenotypes, such as the wrinkled leaf morphology, excessive and partially non geotropic root systems and the ability of leaf explants to differentiate roots in a hormone-free culture medium. Particularly interestingly, most of these traits were shown by plants transformed with a TL-DNA segment encompassing the single ORF 11, corresponding to the rolB locus. Hairy root can be induced by this latter T-DNA segment on wounded stems of tobacco plants; hairy root induction on carrot discs requires, on the contrary, a more complex complement of TL-DNA genes.Abbreviations YMB yeast mannitol broth - MS Murashige and Skoog medium - 6-BAP 6-benzylaminopurine - NAA naphthalene acetic acid - Km kanamycin - Cb carbenicillin     

The role of hormones on morphogenesis of thin layer explants from normal and transgenic tobacco plants

The organogenic potential of thin layer stem explants of non-reproductive tobacco plants was tested on a hormone-free medium and under various hormonal conditions. A comparison was made between thin layers excised from normal and transgenic plants at the same developmental stage. The transgenic plants were transformed by insertion of TR- and TL-DNA from Agrobacterium rhizogenes 1855 root-inducing plasmid. The aim was to identify hormonal conditions capable of stimulating the expression of the flowering competence present in the differentiated stem tissues at the induced stage before any visible sign of transition to reproductive development. Flower neoformation, observed at the end of the culture period (day 25), occurred on untransformed thin layers only with kinetin treatment. Explants from transgenic plants showed flower bud regeneration on hormone-free medium, indoleacetic acid alone (1 μ M ), kinetin alone (1 μ M ), and most abundantly on indoleacetic acid plus kinetin (1 μ M each). No flower formation was observed on indolebutyric acid plus kinetin (10 μ M and 0.1 μ M , respectively) in both normal and transgenic explants. The latter treatment enhanced rooting instead, above all in the transgenic explants. On hormone-free medium vegetative bud formation was well expressed both by untransformed and transgenic explants, and enhanced by the combined, equimolar concentrations of indoleacetic acid and kinetin.
The results show that cytokinin allows flowering in florally determined stem explants from normal plants. In the transgenic explants, the flowering response increases when indoleacetic acid is added to cytokinin, thus suggesting a role for auxin in enhancing the expression of the florally determined state in thin cell layers of non-reproductive plants.     

Inhibition by Ethylene of Auxin-Promotion of Flower Bud Formation in Tobacco Explants Is Absent in Plants Transformed by Agrobacterium rhizogenes

The in vitro regeneration of flower buds was studied in pedicel explants from tobacco (Nicotiana tabacum L., cv Petit Havana) transformed with Agrobacterium rhizogenes, pRi 1855 (agropine type). At a low concentration (0.1 micromolar) of 1-naphthalene-acetic acid, pedicel strips from phenotypically aberrant plants regenerated two to three times more flower buds than explants from untransformed tobacco. Intermediate bud numbers were observed in transformants with a less extreme phenotype. The results can be explained by an increased sensitivity of the transformed explants to auxin with respect to flower bud regeneration. The effect of transformation on the auxin response is fully accounted for by the absence of a negative interaction of endogenous ethylene with 1-naphthaleneacetic acid, a phenomenon normally encountered in untransformed tissues. Three observations led to this conclusion. Application of 1 micromolar AgNO₃ to untransformed explants increased the number of flower buds to the level observed in transformed tissues but had no effect on transformed pedicel strips; exposure to 10 microliters per liter ethylene strongly reduced the response to auxin at all concentrations in untransformed explants but was almost ineffective in the transformed tissues; and endogenous ethylene synthesis occurred at the same rate in both types of explants.     

Crown gall disease and hairy root disease : a sledgehammer and a tackhammer

The neoplastic diseases crown gall and hairy root are incited by the phytopathogenic bacteria Agrobacterium tumefaciens and Agrobacterium rhizogenes, respectively. Although the molecular mechanism of T-DNA transfer to the plant most likely is the same for both species, the physiological basis of tumorigenesis is fundamentally different. Crown gall tumors result from the over-production of the phytohormones auxin and cytokinin specified by A. tumefaciens T-DNA genes. Although the T-DNA of some Riplasmids of A. rhizogenes contains auxin biosynthetic genes, these loci are not always necessary for hairy root formation. Recent experiments suggest that hairy root tumors result from the increased sensitivity of transformed cells to endogenous auxin levels. An understanding of hairy root tumorigenesis will likely result in an increased knowledge of plant developmental processes.     

The role of auxin in hairy root induction

Summary We have investigated the relative role of auxin and of Agrobacterium rhizogenes T-DNA in the induction of hairy roots. By infecting carrot discs with suitably constructed bacterial strains containing different T-DNA complements, we have shown that both auxin and the presence of T-DNA in the carrot cells are required for root growth on the discs. Auxin added alone or in combination with cytokinin is not sufficient to induce rooting on uninfected discs. Also cells transformed by T-DNA containing only auxin synthetic genes very rarely differentiate into roots. On the other hand auxin is necessary for hairy root induction since A. rhizogenes devoid of T-DNA-borne auxin genes is not capable of eliciting symptoms in the absence of hormone. Auxin is not required for either T-DNA transfer or T-DNA expression in the transformed host. Cells infected in the absence of auxin, which do not respond by rooting, do contain T-DNA whose expression is shown by the synthesis of hairy root opines; subsequent addition of auxin to these quiescent transformed cells results in root development. A model for hairy root induction where the action of T-DNA is envisaged as conferring auxin responsiveness to the transformed cells is discussed.     

Hairy Root-activation Tagging: a High-throughput System for Activation Tagging in Transformed Hairy Roots

Activation tagging is a powerful technique for generating gain-of-function mutants in plants. We developed a new vector system for activation tagging of genes in “transformed hairy roots”. The binary vector pHR-AT (Hairy Root-Activation Tagging) and its derivative pHR-AT-GFP contain a cluster of rol (rooting locus) genes together with the right border facing four tandem repeats of the cauliflower mosaic virus (CaMV) 35S enhancer element on the same T-DNA. Transformation experiments using Arabidopsis, potato, and tobacco as model plants revealed that upon inoculating plants with Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants within a short period of time, and root culture lines were subsequently established. Molecular analyses of the pHR-AT-GFP-transformed Arabidopsis lines showed that expression of the genes adjacent to the T-DNA insertion site was significantly increased. This system may facilitate application of the activation-tagging approach to plant species that are recalcitrant to the regeneration of transgenic plants. High-throughput metabolic profiling of activation-tagged root culture lines will offer opportunities for identifying regulatory or biosynthetic genes for the production of valuable secondary metabolites of interest.     

Gain of function assays identify non- rol genes from Agrobacterium rhizogenes TL-DNA that alter plant morphogenesis or hormone sensitivity

This study tested the morphogenetic potential of 15 open reading frames of the TL-DNA of Agrobacterium rhizogenes strain HRI. These open reading frames were expressed individually under the control of the 35S RNA promoter in transgenic tobacco plants ( Nicotiana tabacum L.). Expression of three T-DNA loci, ORF3n, ORF8 and ORF13, alters plant morphogenesis or the response of transgenic tissues to plant hormones. ORF3n transgenic plants are characterized by retarded flowering, altered internode elongation, altered leaf shape and, in particular, leaf tip necrosis. ORF3n and ORF8 expression reduces the sensitivity to auxin and cytokinin in combination or auxin alone. Tetracycline-dependent expression of ORF13 overcomes a selection of low levels of expression during plant regeneration and reveals a strong inhibitory effect of the ORF13 gene product on cell division and cell elongation. We conclude that the A. rhizogenes TL-DNA harbors genetic information that is important for pathogenicity apart from the well studied rol genes. We propose that these genes play mainly a negative regulatory role during pathogenesis. Moreover, these loci might be relevant to successful infections in specific host plants.     

Agrobacterium rhizogenes-mediated transformation of scented geranium

A method is described for producing genetically transformed plants from explants of three scentedPelargonium spp. Transgenic hairy root lines were developed fromPelargonium spp leaf explants and microcuttings after inoculation withAgrobacterium rhizogenes strains derived from the agropine A4 strain. Hairy root lines grew prolifically on growth regulator-free medium. Transgenic shoots were regenerated from hairy roots and the plants have been successfully transferred to soil. The phenotype of regenerated plants has been characterized as having abundant root development, more leaves and internodes than the controls, short internodes and highly branched roots and aerial parts. Southern blot analyses have confirmed the transgenic nature of these plants.     

T-DNA analysis of plants regenerated from hairy root tumors

Summary Plants regenerated from hairy root tumors induced on Nicotiana glauca and Nicotiana tabacum by Agrobacterium rhizogenes strain A4 were examined for the presence of T-DNA. Regenerated N. tabacum plants contained intact copies of both TL-DNA and TR-DNA. However, plants regenerated from N. glauca tumors did not contain the TR-DNA region corresponding to the tms (auxin synthesis) genes. Some of the regenerants exhibited an abnormal phenotype which is characterized by severe leaf wrinkling. This phenotype is correlated with the presence of TL-DNA, but not TR-DNA.     

Salt stress induced soybean <Emphasis Type="Italic">GmIFS1</Emphasis> expression and isoflavone accumulation and salt tolerance in transgenic soybean cotyledon hairy roots and tobacco

Effects of isoflavones on plant salt tolerance were investigated in soybean (Glycine max L. Merr. cultivar N23674) and tobacco (Nicotiana tabacum L.). Leaf area, fresh weight, net photosynthetic rate (Pn), and transpiration rate (Tr) of soybean N23674 plants treated with 80 mM NaCl were significantly reduced, while a gene (GmIFS1) encoding for 2-hydroxyisoflavone synthase was highly induced, and isoflavone contents significantly increased in leaves and seeds. To test the impact of isoflavones to salt tolerance, transgenic soybean cotyledon hairy roots expressing GmIFS1 (hrGmIFS1) were produced. Salt stress slightly increased isoflavone content in hairy roots of the transgenic control harboring the empty vector but substantially reduced the maximum root length, root fresh weight, and relative water content (RWC). The isoflavone content in hrGmIFS1 roots, however, was significantly higher, and the above-mentioned root growth parameters decreased much less. The GmIFS1 gene was also transformed into tobacco plants; plant height and leaf fresh weight of transgenic GmIFS1 tobacco plants were much greater than control plants after being treated with 85 mM NaCl. Leaf antioxidant capacity of transgenic tobacco was significantly higher than the control plants. Our results suggest that salt stress-induced GmIFS1 expression increased isoflavone accumulation in soybean and improved salt tolerance in transgenic soybean hairy roots and tobacco plants.     

CIPK6, a CBL-interacting protein kinase is required for development and salt tolerance in plants

Calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK) mediate plant responses to a variety of external stresses. Here we report that Arabidopsis CIPK6 is also required for the growth and development of plants. Phenotype of tobacco plants ectopically expressing a homologous gene ( CaCIPK6 ) from the leguminous plant chickpea ( Cicer arietinum ) indicated its functional conservation. A lesion in AtCIPK6 significantly reduced shoot-to-root and root basipetal auxin transport, and the plants exhibited developmental defects such as fused cotyledons, swollen hypocotyls and compromised lateral root formation, in conjunction with reduced expression of a number of genes involved in auxin transport and abiotic stress response. The Arabidopsis mutant was more sensitive to salt stress compared to wild-type, while overexpression of a constitutively active mutant of CaCIPK6 promoted salt tolerance in transgenic tobacco. Furthermore, tobacco seedlings expressing the constitutively active mutant of CaCIPK6 showed a developed root system, increased basipetal auxin transport and hypersensitivity to auxin. Our results provide evidence for involvement of a CIPK in auxin transport and consequently in root development, as well as in the salt-stress response, by regulating the expression of genes.     

Characterization of ornamental Datura plants transformed by Agrobacterium rhizogenes

Summary Datura arborea and D. sanguinea hairy roots were produced by cocultivation of leaf fragments with Agrobacterium rhizogenes strain NCPP 1855. Adventitious buds emerged spontaneously, without exogenous growth regulators, from seven hairy root clones of D. arborea and from one hairy root clone of D. sanguinea. Regenerated plants were successfully acclimatized in the greenhouse. The integration of the bacterial TL-DNA into the genome of the putative transformed plants was confirmed by Southern blot analysis. Transgenic plants displayed increased ability to root in vivo. Morphological traits with relevant ornamental value like plant height, leaf number, size and shape, internode number, and internode length were also affected. Transformation by wild-type Ri TL-DNA provided the chance to study plant growth and differentiation and to select improved genotypes.     

Virulence properties of strains of agrobacterium on the apical and Basal surfaces of carrot root discs

Most pathogenic strains of Agrobacterium are able to induce crown gall or hairy root on both the apical surface (facing the root tip) and the basal surface (facing the shoot) of carrot (Daucus carota L.) root discs. Tumorigenic strains carrying mutations in the shoot inhibition region of the T-DNA (TL-DNA genes 1 and 2) are markedly attenuated on the basal surface but remain virulent on the apical surface. Coinoculation of two attenuated tumorigenic strains, with mutations in gene 1 and gene 2, respectively, resulted in restoration of virulence on the basal surface. Wild type hairy root-inducing strains can be divided into two groups: those that are virulent on both apical and basal surfaces and those that are virulent only on the apical surface. α-Naphthalene acetic acid stimulated virulence of hairy root strain TR7, belonging to the latter group, on the basal surface. Attenuated virulence on the basal surface can be explained in terms of an auxin deficiency in the basal tissues and unidirectional auxin transport to the apical surface.     

Production of ROL gene transformed plants of Rosa hybrida L. and characterization of their rooting ability

Transgenic plants of the rootstock Rosa hybrida L. cv. Moneyway were produced via a two-step procedure. First, kanamycin-resistant roots were generated on stem slices from micropropagated shoots, which were cocultivated with Agrobacterium tumefaciens containing the neomycin phosphotransferase II (NPTII) gene for conferring kanamycin resistance, together with individual ROL genes from A. rhizogenes. Root formation was quite efficient and up to two kanamycin-resistant roots per stem slice were produced. In the second step, these roots were used to regenerate transgenic plants via somatic embryogenesis. Although regeneration lasted up to 12 months, production of several transformants was successfully accomplished. Untransformed escapes were not found, indicating that the initial selection on kanamycin resistance was reliable.The presence of a combination of ROLA, B and C genes enhanced adventitious root formation on micropropagated shoots and explants of stems and leaves. It appears that the auxin sensitivity was increased to such a degree that cells were able to respond even to endogenous auxins present in shoots and leaves. Rooting experiments in greenhouse demonstrated that adventitious root formation on cuttings was improved threefold upon introduction of these ROL genes. It is concluded that a method was developed for the production of ROL gene transformed roses with improved rooting characteristics.     

Rose-scented geranium (<Emphasis Type="Italic">Pelargonium</Emphasis> sp.) generated by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated Ri-insertion for improved essential oil quality

Transgenic plants of rose-scented geranium (Pelargonium graveolens cv. Hemanti) have been produced from Agrobacterium rhizogenes (strains A₄ and LBA9402) mediated hairy root cultures. Amongst the explants tested, leaves were most responsive followed by the petioles and internodal segments, respectively. The A₄ strain performed better for all the three explants both in terms of frequency of response and time requirement for hairy root induction. Transgenic shoots could be obtained by spontaneous regeneration without intervening callus phase amongst 16% and 12% root lines of A₄ and LBA 9402 origin, respectively, or they were induced in 29% and 22% hairy root lines of A₄ and LBA9402 origin, respectively, with different hormonal supplementation. These transgenic plants showed 30% survival as against 90% of their control under the confined environment of glasshouse. The transgenic plants were of similar morphotype having increased branching, higher number of leaves with increased dentations, short and round stature, highly branched root system and absence of leaf wrinkling. These transgenic plants showed opine positive results even after 5 months of their transfer to the glasshouse. The essential oil compositions of 81% of these transgenics were qualitatively similar to that of the wild type parent. However, two transgenic plants (LZ-3 and 14TG) showed increase in concentrations of geraniol and geranyl esters signifying improved oil quality with respect to the citronellol:geraniol ratio. These two oils having better olfactory value represent an improvement over that of the wild type parent from the commercial point of view.     

Independent induction of transformed roots by the TL and TR regions of the Ri plasmid of agropine type Agrobacterium rhizogenes

Summary To analyse the respective role of TL- and TR-DNA in root induction by agropine-type Agrobacterium rhizogenes Ri plasmids, deletions covering the TL- or the TR-regions were constructed in vitro and introduced into pRiA4 by marker exchange. Each T-region of pRiHRI was also cloned separately on an independent replicon and used in a binary system with the virulence functions of either an Ri or a Ti plasmid provided in trans. Transformed roots were induced on tobacco and tomato explants by TL-DNA as well as by TR-DNA, suggesting that agropine type Ri plasmids from strains A4 and HRI can induce root proliferation by two independent transformation mechanisms. The root induction by the TR-DNA is probably due to auxin biosynthesis by gene products of aux loci homologous to the tms genes of Ti plasmid T-DNA. The molecular mechanism of root proliferation induced by the TL-DNA is probably equivalent to that of mannopine type Ri plasmid T-DNA.     

Structure and function of root-inducing (Ri) plasmids and their relation to tumor-inducing (Ti) plasmids

The abilities of Agrobacterium tumefaciens and A. rhizogenes to transform dicotyle-dons and cause crown gall and hairy root disease are caused by the presence of tumor inducing (Ti) and root inducing (Ri) plasmids. During transformation plasmid T-DNA (transferred DNA) is inserted into the plant genome. The T-region is flanked by 25 bp direct repeats, which are essential for transfer. The T-regions contain oncogenes that are expressed in the plants. Some of these code for enzymes that synthesize auxin or cytokinin. Another type, present in Ri plasmids only, appears to impose a high hormone sensitivity on the infected tissue. The T-DNA also contains genes for enzymes synthesizing opines, which the bacteria catabolize. The T-DNA transfer is initiated by the induction of genes in the virulence (vir) region of the plasmid by phenolic compounds secreted by wounded tissue. The products of the vir -genes and of chromosomal genes mediate transfer of T-DNA to the plant cells. Crown gall disease is caused by production of auxin and cytokinin by the transferred T-DNA. The T-DNA of Ri plasmids codes for at least three genes that each can induce root formation, and that together cause hairy root formation from plant tissue. Current results indicate that the products of these genes induce a potential for increased auxin sensitivity that is expressed when the transformed cells are subjected to a certain level of auxin. After this stage the transformed roots can be grown in culture without exogenous supply of hormones.     

Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens -transformed roots and Agrobacterium rhizogenes-transformed hairy roots

Medicago truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes.